Microarrays are now a commonly used format for most high-throughput screening applications in genomics, proteomics and glycomics and the ability to analyse them by more than a single readout method is desirable to broaden their applications. While the first examples to couple affinity-chromatography with MALDI-Tof MS employed off-line isolation of analytes onto blotting membranes or functionalized beads, the advantages of directly functionalizing the MALDI sample plate were rapidly recognized. The covalent attachment of capture molecules to the sample plate has allowed the observation of antibody-antigen, protein-protein, lectin-carbohydrate interactions or the sequencing of captured oligonucleotides by MALDI-Tof MS. With a cleavable linkage to the surface, any enzymatic or chemical transformation leading to a mass change on the immobilized substrates can be observed directly by MALDI-Tof MS. Detachment of the immobilized analytes from the sample plate under laser irradiation have been achieved via photocleavable linkers, thiolates that desorb under laser irradiation or non-covalent fluorous and hydrophobic interactions.
However, the mass spectrometric analysis of microarrays, particularly those with small spot sizes (e.g., between 100-600 μm in diameter) has been hampered by constraints in method sensitivity and fabrication and efforts in this direction were limited to commercial well plate formats or slides with manually-deposited binding agent.
Glycan microarrays have advanced our understanding of carbohydrate-protein interaction and the associated field of glycomics like no other high-throughput technology. Thousands of individual carbohydrate binding events can be observed at the same time with often attomolar receptor sensitivity and, perhaps more important, using only picomolar amounts of glycan ligands. Most glycan array studies have focused on the analysis of substrate specificities of carbohydrate binding proteins, the study of cell surface receptors in bacteria and eukaryotic cell lines or the measurement of substrate specificities of selected glycosyltransferases employing fluorescent readout methods.
The on-chip activity screening of carbohydrate processing enzymes (glycosyltransferases, hydrolases, transglycosidases etc.) has important applications like the discovery of new glycosyltransferases, the evaluation of substrate specificities of biomass degrading enzymes, specificities of carbohydrate modifying enzymes like sulfatases, acylases, kinases etc., the rapid screening of potential inhibitors against a specific enzyme target or monitoring the outcome of on-chip enzymatic synthesis. In these cases, the use of fluorescently tagged probes or substrates only provides qualitative information and, furthermore, a ranking of ligands is often compromised by low individual binding strengths between ligand and affinity probe.
Several groups have picked up on the pioneering work of Mrksich (Su and Mrksich, 2002) to analyse surface bound glycans and other biomolecules by MALDI-TOF mass spectrometry. Due to constrains in fabrication and method sensitivity these surface-based mass spectrometry studies were carried out at the macroscopic scale using commercial samples with well dimensions of around 5 mm or manually spotted larger spots (Su and Mrksich, 2002; Sanchez-Ruiz et al., 2011; Chang et al. 2010). Microarrays, however, present spot sizes at least 10 times smaller in the range of 200-800 μm, which are resolved with a resolution of 5-10 μm by a standard slide scanner.
Sanchez-Ruiz et al. 2011 describes the use of MALDI-Tof mass spectroscopic analysis of enzyme activity using glycan arrays formed gold surfaces using a method which involves initially functionalising glycans with lipid tags and then non-covalently immobilising them on alkylthiolate SAM on a MALDI-Tof plate. Glycan arrays made using a similar approach are disclosed in Beloqui et al. 2012 and used for screening glycosidase specificity in environmental samples.
US Patent Application No: 2010/0004137 (Mrksich et al.) describes biochips with gold surfaces on which self-assembled monolayers (SAMs) of alkanethiol molecules are covalently attached via thiol chemistry. Glycans are immobilised on the biochips through reaction with terminal oligo(ethylene glycol) groups provided on the alkanethiol molecules so that they become covalently linked to the biochip and studied using MALDI-Tof spectroscopy.
Chang et al. 2010 makes glycan arrays in which the glycans are covalently or non-covalently attached to aluminium oxide coated glass (ACG) slides. For non-covalent attachment, the glycans are initially tagged with polyfluorinated hydrocarbon tails and then spotted robotically onto the ACG slide surface containing a layer of polyfluorinated hydrocarbon terminated with phosphonate. For covalent attachments, the glycans with a phosphonic acid tail were synthesized and spotted robotically onto the ACG slide surface. The non-covalent array was characterized by MS-Tof via ionization/desorption at a low laser energy without addition of matrix.
There remains a need in the art to further develop microarrays, and in particular to develop efficient ways of making arrays and/or to develop arrays capable of providing multimodal readout.